Differences in the intracellular distribution of acid-sensitive doxorubicin-protein conjugates in comparison to free and liposomal formulated doxorubicin as shown by confocal microscopy

Purpose. To investigate differences in the cellular uptake and intracellula r distribution of protein-bound doxorubicin in comparison to free doxorubic in and a liposomal formulation (CAELYX(R)). Methods. LXFL 529 lung carcinoma cells were incubated with an acid-sensitiv e transferrin and albumin conjugate of doxorubicin, a stable albumin doxoru bicin conjugate, and free and liposomal doxorubicin for up to 24 h. The upt ake of doxorubicin was detected with confocal laser scanning microscopy (CL SM). To investigate the intracellular localization of the anticancer drug, lysosomes, Golgi apparatus, and mitochondria were also stained by various o rganelle-specific fluorescent markers. In vitro efficacy of the doxorubicin derivatives was examined with the BrdU incorporation assay. Results. The acid-sensitive albumin and transferrin doxorubicin conjugates showed enhanced cytotoxicity in comparison to liposomal doxorubicin, wherea s the stable albumin-doxorubicin conjugate showed only marginal activity. O f all compounds tested, doxorubicin showed the highest cytotoxicity. CLSM s tudies with specific markers for lysosomes, mitochondria, and the Golgi app aratus demonstrated that protein-bound doxorubicin or liberated doxorubicin was accumulated in the mitochondria and Golgi compartments, but not in the lysosomes after 24 h. Free doxorubicin showed a time-dependent intracellul ar shift from the nucleus to the mitochondria and Golgi apparatus. Fluoresc ence resulting from incubation with CAELYX was primarily detected in the nu cleus. Conclusions. Our results indicate that other organelles in addition to the cell nucleus are important sites of accumulation and interaction for protei n-bound doxorubicin or intracellularly released doxorubicin as well as for free doxorubicin.